Impact of measurable residual disease in combination with CD19 on postremission therapy choices for adult t(8;21) acute myeloid leukemia in first complete remission

Abstract Background The post‐remission therapy (PRT) choices for adult t(8;21) acute myeloid leukemia (AML) in first complete remission (CR1) need to be further explored. Aims We aimed to investigate the impact of measurable residual disease (MRD) combined with CD19 on PRT choices for adult t(8;21) AML in CR1. Methods A total of 150 t(8;21) AML patients were enrolled, including 67 underwent chemotherapy (CMT) and 83 allogeneic hematopoietic stem cell transplantation (allo‐SCT) as PRT in CR1. Subgroup analyses were performed according to MRD level after three cycles of chemotherapy combined with CD19 expression. Results Multivariate analysis indicated MRDhigh after three courses of treatment (HR, 0.14 [95% CI, 0.03–0.66]; p = 0.013) and CD19 negativity (HR, 0.14 [95% CI, 0.02–0.96]; p = 0.045) were risk factors for relapse, while allo‐SCT was protective factor for relapse (HR, 0.34 [95% CI, 0.15–0.75]; p = 0.008). Grouped by MRD after three courses of chemotherapy, allo‐SCT had lower CIR (p < 0.001) and better OS (p = 0.003) than CMT for MRDhigh patients, CMT showed a higher CIR (35.99% vs. 15.34%, p = 0.100) but comparable OS (p = 0.588) than allo‐SCT for MRDlow patients. Grouped by CD19 expression, allo‐SCT demonstrated lower CIR (p < 0.001) and better OS (p = 0.002) than CMT for CD19− patients. CMT had a higher CIR (41.37% vs. 10.48%, p = 0.007) but comparable OS (p = 0.147) than allo‐SCT for CD19+ patients. Grouped by MRD combined with CD19, MRDhigh/CD19+ subsets were identified out of CD19+ patients benefiting from allo‐SCT with lower CIR (p = 0.002) and superior OS (p = 0.020) than CMT. CMT preserved comparable CIR (p = 0.939) and OS (p = 0.658) with allo‐SCT for MRDlow/CD19+ patients. MRDlow/CD19− subsets were also identified from MRDlow patients requiring allo‐SCT with lower CIR (p < 0.001) and superior OS (p = 0.008) than CMT. Allo‐SCT maintained lower CIR (p < 0.001) and superior OS (p = 0.008) than CMT for MRDhigh/CD19− patients. Conclusions MRD combined with CD19 might optimize PRT choices for adult t(8;21) AML patients in CR1.

In the past, researchers have used real-time quantitative polymerase chain reaction (RT-qPCR) methodology to routinely monitor the RUNX1/RUNX1T1 transcript levels as measurable residual disease (MRD).The MRD assessment in t(8;21) AML patients helps in identifying the patients with a higher relapse risk. 6,7As a result, it was employed to guide PRT for t(8;21) AML.However, the optimal time point and the threshold levels of RUNX1/RUNX1T1 transcripts that indicate relapse are still debatable. 9,10Furthermore, t(8;21) AML patients with a low MRD level after chemotherapy, such as >3 log reduction of RUNX1/RUNX1T1 transcripts in comparison to their pretreatment baseline level, exhibited a cumulative incidence of relapse (CIR) of up to 30%. 7,9herefore, MRD alone might not be enough for riskdirect PRT selection.Recently, Qin et al. investigated the impact of c-Kit mutation in combination with MRD on outcomes in t(8;21) AML. 11They observed that the KITD816/D820 patients with higher MRD levels after the second consolidation treatment showed the maximal 3-year CIR.On the other hand, the patients undergoing allo-SCT had significantly lower CIR and higher overall survival (OS) compared with those undergoing chemotherapy.They further noted that the KITD816/ D820 patients with lower MRD levels, KITN822 patients, and KITexon8/WT patients with higher MRD levels displayed intermediate 3-year CIR, while those undergoing allo-HSCT had lower CIR but comparable OS in comparison to patients undergoing chemotherapy.
It was also noted that the KITexon8/WT patients with lower MRD levels demonstrated the lowest 3-year CIR, wherein allo-SCT decreased both relapse and survival. 11herefore, integrated evaluation of baseline prognosticator and MRD might better guide risk stratification and PRT in t(8;21) AML.
6][17] However, it is not clear whether the combination of CD19 and MRD could better guide PRT choices.In this study, we retrospectively analyzed two different postremission approaches with allo-SCT or consolidation chemotherapy (CMT) for t(8;21) AML in CR1, to determine whether the combination of CD19 and MRD could optimize the PRT choices for t(8;21) AML patients in CR1.

| Patients
In this study, we screened a total of 176 recently diagnosed t(8;21) AML patients at Nanfang Hospital (Guangzhou province, China) between January 2010 and June 2021.Depending on their PRT approaches, the patients were categorized into two groups, that is, those undergoing consolidation chemotherapy (CMT) and those undergoing allo-SCT.The CMT group included patients who had undergone at least two consolidation chemotherapy cycles and were not scheduled for upfront SCT.Furthermore, the patients in the CMT group also included the patients who had relapsed after CMT and were administered SCT subsequently.The following inclusion criteria were used for enrolling the patients: (1) aged above 14 years, (2) newly diagnosed with AML with t(8;21) and/or RUNX1/ RUNX1T1 transcripts, and (3) those who acquired CR after 1-2 induction chemotherapy cycles.On the other hand, the following exclusion criteria were implemented in this study: (1) patients who died during induction therapy, (2) those who could not achieve CR after two induction allo-SCT with lower CIR (p < 0.001) and superior OS (p = 0.008) than CMT.Allo-SCT maintained lower CIR (p < 0.001) and superior OS (p = 0.008) than CMT for MRD high /CD19 − patients.chemotherapy cycles, (3) those who received less than two consolidation cycles, and (4) the patients who did not receive any form of treatment.All the surviving patients were last followed up until December 31, 2021.The institutional review board at the Nanfang Hospital reviewed and approved the procedures implemented in this study.In accordance with the principles laid down in the Declaration of Helsinki, all the patients or their guardians were asked to provide their written informed consent before enrolling in this study.

MRD assessment
Here, we used the Giemsa and reverse banding techniques and the fluorescence in situ hybridization procedure for carrying out cytogenetic analyses.Furthermore, the molecular markers used in the study were identified using the polymerase chain reaction (PCR) and 167-gene next-generation sequencing (NGS) (Table S1).The bone marrow specimens were collected from the enrolled patients at different time points, that is, after induction, after each PRT cycle, every 2 months in Year 1, every 3 months in Year 2, every 4 months in Year 3, and every 6 months in Years 4 and 5. 18 We used the real-time quantitative reverse transcription PCR (RT-PCR) technique for quantifying the RUNX1/RUNX1T1 transcript levels in the samples, and the results were presented in the form of the RUNX1/RUNX1T1/ABL ratio. 9After three courses of chemotherapy, 3-log reduction in RUNX1/RUNX1T1 transcripts (0.1%) compared with the pretreatment baseline of 100% in our center, was used as a threshold to distinguish high MRD level (MRD high ≥ 0.1%) from low MRD level (MRD low < 0.1%).

| Treatment protocols
The regimens that were used for induction therapies included the administration of either daunorubicin (60 mg/ m 2 ) or idarubicin (12 mg/m 2 ) for three continuous days in combination with cytarabine (100 mg/m 2 ) for 7 days (i.e., "3 + 7" regimen).In the case of patients who could not achieve CR after their first induction, a second induction was administered involving daunorubicin (60 mg/ m 2 ) or idarubicin (10 mg/m 2 ) every day, for 3 days, combined with cytarabine (2.0 g/ m 2 ) every 12 h on Days 1-3 (i.e., "3 + 3" regimen), or the same regimen that was administered during the first induction cycle.After achieving the first CR, MRD high patients (RUNX1/RUNX1T1/ABL ≥ 0.1%) after three courses of chemotherapy were recommended for allo-HSCT, while MRD low patients (RUNX1/RUNX1T1/ ABL < 0.1%) after three courses of chemotherapy were recommended for cytarabine-based consolidation chemotherapy.Among patients who could not comply with the treatment recommendations described above due to patient bias or donor availability, MRD low patients underwent allo-HSCT and MRD high patients underwent chemotherapy as postremission treatment.The cytarabine-based consolidation therapy included the "3 + 3" regimen and an intermediate/high-dose of cytarabine (2-3 g/m 2 ) twice each day on Days 1-3. 19Allo-SCT was also advised for patients who had relapsed after CMT. 17 The donor selection principle for allo-SCT was based on the Chinese consensus. 20,21All the patients received busulfan-based myeloablative conditioning regimens.

| Evaluation points and definition
The basic objective of this research study was to determine the 3-year OS duration of the patients.The second endpoint included the cumulative incidence of relapse (CIR), leukemia-free survival (LFS), nonrelapse mortality (NRM), GVHD-free survival, and relapse-free survival (GRFS).Complete Remission (CR) was defined as <5% blasts based on the morphologic assessment of the bone marrow (BM) samples with no clear evidence that indicates dysplasia in BM and no development of leukemia outside the hematopoietic system.Relapse was defined based on the morphologic evidence noted in the marrow, peripheral blood, or extramedullary regions. 1 NRM was described as death without any evidence indicating the relapse of leukemia.OS was calculated from Day 1 of the therapy to death or censored at the final follow-up.Furthermore, we determined the LFS from CR1 to relapse or death or censored at the final followup.The pattern and severity of organ involvement were used to define and grade acute GVHD, while the NIH criteria were used for defining and grading chronic GVHD. 22raft-vs-host-disease-free, relapse-free survival (GRFS) events were defined as grade III or IV acute GVHD, chronic GVHD requiring systemic immunosuppressive treatment, leukemia relapse, or death from any cause from CR1 to last follow-up.The GRFS condition presented by the patients in the chemotherapy group was regarded as equal to LFS as they did not show any incidence of GVHD and hence was compared to that exhibited by the patients in the allo-SCT group.

| Statistical analysis
The differences in the data among different groups were compared using the χ 2 test or Fisher's exact test for categorical variables, whereas the Mann-Whitney U test was employed for comparing the continuous variables.The Kaplan-Meier technique was utilized for estimating the OS and LFS and compared with the log-rank test.Cumulative incidence curves were used in a competing risk setting with relapse treated as a competing event to calculate NRM and with NRM treated as a competing risk to calculate relapse.The Cox regression model was implemented for conducting the univariate and multivariate analysis.The multivariate analysis included the variables showing p < 0.10 in univariate analysis or any variable affecting the outcome.A two-sided p-value of <0.05 was considered statistically significant.The data in the study were statistically analyzed using SPSS (ver.26.0) and R (4.1.1)software.

| PRT selection and outcomes based on MRD
In the case of t(8;21) AML patients with MRD high after three cycles of treatment, it was noted that the patients in the allo-SCT group displayed a significantly lower 3-year CIR compared to the patients in CMT (10.03% vs. 74.31%,p < 0.001).They further exhibited better LFS in comparison to the CMT group (82.11% vs. 24.90%,p < 0.001).Furthermore, the Allo-SCT group also showed better 3-year GRFS (52.56% vs. 24.90%,p = 0.009) and 3-year OS (85.58% vs. 56.03%,p = 0.003) than the CMT patients (Figure 2A).On the contrary, in the case of MRD low patients after three cycles of treatment, those in the allo-SCT preserved a trend toward lower 3-year CIR (15.34% vs. 35.99%,p = 0.100) compared to the CMT group.However, patients in allo-SCT and CMT groups exhibited similar 3-year LFS (74.35% vs. 61.55%,p = 0.274), 3-year GRFS (52.87% vs. 61.55%,p = 0.576), and 3-year OS (80.85% vs. 77%, p = 0.588) values (Figure 2B).This study also indicated allo-SCT could reduce the relapse rate and improve the survival duration of the MRD high patients compared to those receiving CMT treatment.However, the 3-year CIR remained at 35.99% in MRD low patients undergoing CMT.
To further figure out whether the predicted power of MRD combined with CD19 was superior to MRD alone for outcomes in t(8;21) AML patients, we utilized timedependent ROC and Harrell's Harmony Index (C-index).Based on the prognosis-related independent factors screened by the multivariate Cox regression, we constructed an OS-related model that included MRD alone or MRD combined with CD19, namely MRD model and MRD/CD19 model.Compared with the MRD model, the MRD/CD19 model had a higher C-index of predicting overall survival (OS) (0.756 vs. 0.671, p = 0.010).For MRD and MRD/CD19 models, the AUC values of 3-year OS were 0.723 and 0.804, 5-year OS were 0.640 and 0.840, respectively (Supplemental figure2).These results suggest that MRD combined with CD19 was more effective than CD19 alone predicting survival.

| DISCUSSION
To our knowledge, this study first demonstrated MRD in combination with CD19 might better guide PRT choices compared with MRD or CD19 alone for t(8;21)AML in CR1.The results showed that for MRD high /CD19 − , MRD high /CD19 + , and MRD low /CD19 − patients, allo-SCT might be recommended in CR1; for MRD low /CD19 + patients, CMT might be recommended.
Optimizing postremission therapy for t(8;21) AML patients remains a major clinical challenge.Several studies have concluded that MRD persistence was related to an increasing relapse risk, resulting in poor outcomes in t(8;21) AML patients. 9,10,23][26] In their studies, Yin et al. and Rücker et al. reported that <3 log reduction of RUNX1/RUNX1T1 transcripts in comparison to the pretreatment baseline values after three courses of chemotherapy indicated a higher relapse risk. 6,9We recently reported that the favorablerisk MRD-positive patients after three chemotherapy cycles or recurrent MRD showed higher CIR and benefited from allo-SCT. 19In this study, MRD high after three courses of treatment was identified as an independent risk factor for relapse.Hence, MRD level after three cycles of treatment was employed to assess PRT choice.Similar to the results reported earlier, 8 we also noted that the t(8;21) patients with MRD high after three courses of treatment benefited significantly from allo-SCT and showed lower CIR and better OS values compared to those undergoing CMT.However, in the case of MRD low patients who were administered three courses of treatment, chemotherapy showed a higher CIR of 35.99%.This finding was similar to the value reported earlier, that is, 30%. 7,9In addition to MRD itself, unfavorable molecular prognostic factors may also affect the rate of relapse in this population.Although allo-SCT tends to decrease relapse when compared to consolidation chemotherapy for MRD low patients after three courses of treatment, the OS and GRFS values were similar in both groups.This could be attributed to the fact that allo-SCT could be used as a salvage treatment strategy after relapse, which was consistent with the findings published in the past. 27,280][31] This gives rise to the question: whether it is possible to identify the subsets in t(8;21) AML patients with low MRD levels that could benefit from allo-SCT in CR1 by integrating other prognostic factors with MRD.
Numerous factors exert an impact on clinical outcomes of t(8;21) AML.These factors mainly consist of cytogenetic and molecular alterations, such as co-occurring gene mutations of tyrosine kinase pathway (such as KIT, N/KRAS, and FLT3), epigenetic regulators and cohesin complex, and additional chromosomal abnormalities. 32,33However, their prognostic significance remained debatable.For example, loss of a sex chromosome, 34,35 additional three or more chromosomal abnormalities, 2,36 and KIT mutations, have been differently reported. 37,386][17] Similarly, our study also revealed that the CD19 − patients showed a higher CIR than CD19 + patients.To date, there have been no reports investigating the impact of CD19 on PRT choices for t(8;21) AML patients.In our study, we observed that allo-SCT significantly decreased the relapse rate and improved the survival duration for CD19 − patients compared with consolidation chemotherapy.For CD19+ patients, allo-SCT had higher LFS than CMT owing to significantly lower CIR in the former, indicating that allo-SCT exerts a stronger anti-leukemia effect than CMT for this subset.However, OS and GRFS were comparable between the two groups.Notably, the 3year CIR remained 41.37% in CD19 + patients undergoing CMT as PRT, indicating CD19 alone might be insufficient to guide PRT selection.
Several studies have demonstrated that the combination of cytogenetic classification and MRD status could help AML patients in CR1 make better PRT choices.Favorable-risk and intermediate-risk patients with high MRD levels benefited from more intensified treatment including allo-SCT. 40,41Qin et al. observed that the integrated evaluation of C-Kit and MRD could better guide risk stratification and PRT in t(8;21) AML patients. 11his study attempted to explore whether the combination of MRD and CD19 was superior to MRD or CD19 alone in guiding PRT.In comparison to CD19 alone, CD19 combined with MRD identified MRD high /CD19 + subsets out of CD19 + patients that benefited from allo-SCT.CMT preserved its advantage over allo-SCT for MRD low /CD19 + patients.Furthermore, in comparison to MRD alone, CD19 combined with MRD also identified MRD low /CD19 − subsets out of MRD low patients that required allo-SCT treatment.Allo-SCT maintained its superiority over CMT for MRD high /CD19 − patients.Therefore, we concluded that CD19 combined with MRD helps optimize PRT choices for t(8;21) AML in CR1.
The lower NRM (9.6%) of allo-SCT group in this study was comparable with our former studies (10.4% and 11.6%, respectively) 19,42 and others' reports (9%). 43This improvement likely results from the accumulation of many individually incremental advances in conditioning therapy, GVHD prophylaxis, modern antiviral and antifungal prophylaxis, infection control, and supportive care. 44The lower median age of 32 years and favorable-risk patient selection in this study might also contribute to the low rate of nonrelapse mortality.
To conclude, we noted that MRD combined with CD19 might better guide PRT choices than MRD or CD19 alone for t(8;21) AML patients in CR1.As a limited number of patients were included in the different subgroups, the results have to be explained cautiously.Additionally, the retrospective bias was inevitable.In the future, we will conduct a multicenter, prospective clinical trial to address the above issues and validate the findings noted above.

F I G U R E 1
The flow diagram of t(8;21) AML patients.CR1, first complete remission; CMT, chemotherapy; Allo-SCT, allogeneic hematopoietic stem cell transplantation.

3. 5 |
PRT selection and outcomes based on MRD in combination with CD19 Finally, we investigate the association of integrated results of CD19 and MRD after three cycles of treatment, PRT selection, and outcomes.In this study, 133 patients were further categorized into four subgroups: (I) subgroup A, MRDhigh/ CD19-; (II) subgroup B, MRDhigh/CD19+; (III) subgroup C, MRDlow/CD19-; and (IV) subgroup D, MRDlow/ CD19+.The patient's characteristics among the four groups are shown in Table
T A B L E 1T A B L E 2 Univariate and multivariate analysis for OS, CIR, and LFS.Abbreviations: Allo-SCT, allogeneic hematopoietic stem cell transplantation; BM, bone marrow; CMT, chemotherapy; CR, complete remission; CIR, cumulative incidence of relapse; LFS, leukemia-free survival; OS, overall survival; WBC, white blood cells.